Activity Of Cathepsin Research Articles

Immortalized Alzheimer’s Disease Astrocytes: Characterization of Their Proteolytic Systems

Alzheimer’s disease (AD) is a progressive neurodegeneration with dysfunctions in both the ubiquitin–proteasome system (UPS) and autophagy. Astroglia participation in AD is an attractive topic of research, but molecular patterns are partially defined and available in vitro models have technical limitations. Immortalized astrocytes from the hippocampus of 3xTg-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively) have been obtained as an attempt to overcome primary cell line limitations and this study aims at characterizing their proteolytic systems, focusing on UPS and autophagy. Both 26S and 20S proteasomal activities were downregulated in 3Tg-iAstro, in which a shift in catalytic subunits from constitutive 20S proteasome to immunoproteasome occurred, with consequences on immune functions. In fact, immunoproteasome is the specific complex in charge of clearing damaged proteins under inflammatory conditions. Parallelly, augmented expression and activity of the lysosomal cathepsin B, enhanced levels of lysosomal-associated membrane protein 1, beclin1, and LC3-II, together with an increased uptake of monodansylcadaverine in autophagic vacuoles, suggested autophagy activation in 3Tg-iAstro. The two proteolytic pathways were linked by p62 that accumulated in 3Tg-iAstro due to both increased synthesis and decreased degradation in the UPS defective astrocytes. Treatment with 4-phenylbutyric acid, a neuroprotective small chemical chaperone, partially restored proteasome and autophagy-mediated proteolysis in 3Tg-iAstro. Our data shed light on the impaired proteostasis in 3Tg-iAstro with proteasome inhibition and autophagic compensatory activation, providing additional validation of this AD in vitro model, and propose a new mechanism of action of 4-phenylbutyric acid in neurodegenerative disorders.

Demonstration, using novel supported planar endosomal membranes (SPEM), that further cathepsin action augments the fusion activity of the primed (19-kDa) Ebola virus glycoprotein.

Ebola virus (EBOV) causes a hemorrhagic fever associated with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes where its glycoprotein (GP) is processed to a 19-kDa form, allowing the glycoprotein to bind to its intracellular receptor Niemann Pick type C1. We previously showed that the cathepsin protease inhibitor, E64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs) and fusion of fluorescent (pseudo)virus particles is monitored by TIRF microscopy. We validated the system by demonstrating the pH dependencies of influenza virus HA- and Lassa virus GP-mediated fusion. Using SPEMs, we next showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH and enhanced by Ca2+, consistent with other studies. We further showed that addition of cathepsins (somewhat more prominently with cathepsin B than L) augments both hemi- and full fusion. Subsequently we found that SPEMs appear to retain cathepsin activity, and that E64d inhibits both hemi- and full fusion mediated by 19-kDa GP. Hence we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of pre-primed 19-kDa Ebola GP. Thus we have provided new evidence for how Ebola GP mediates fusion with endosomes, and we have developed a novel approach employing SPEMs that can now be used for studies of any virus that fuses in endosomes.

In silico insights into procathepsin S maturation mediated by glycosaminoglycans

Procathepsins, inactive precursors of cathepsins are present in the extracellular matrix (ECM) and in lysosomes. Their active forms are involved in a number of biologically relevant processes, including bone resorption, intracellular proteolysis and regulation of programmed cell death. These processes might be mediated by glycosaminoglycans (GAGs), long unbranched periodic negatively charged polysaccharides. GAGs are also present in ECM and play important role in anticoagulation, angiogenesis and tissue regeneration. GAGs not only mediate the enzymatic activity of cathepsins but can also regulate the process of procathepsin maturation, as it was shown for procathepsin B and S. In this study, we propose the molecular mechanism underlying the biological role of GAGs in procathepsin S maturation and compare our findings with computational data obtained for procathepsin B. We rigorously analyse procathepsin S–GAG complexes in terms of their dynamics, free energy and potential allosteric regulation. We conclude that the GAG binding region might have an effect on the dynamics of procathepsin S structure and so affect its maturation by two different mechanisms.

Chloroquine Enhances Death in Lung Adenocarcinoma A549 Cells Exposed to Cold Atmospheric Plasma Jet.

Cold atmospheric plasma (CAP) is an intensively-studied approach for the treatment of malignant neoplasms. Various active oxygen and nitrogen compounds are believed to be the main cytotoxic effectors on biotargets; however, the comprehensive mechanism of CAP interaction with living cells and tissues remains elusive. In this study, we experimentally determined the optimal discharge regime (or semi-selective regime) for the direct CAP jet treatment of cancer cells, under which lung adenocarcinoma A549, A427 and NCI-H23 cells demonstrated substantial suppression of viability, coupled with a weak viability decrease of healthy lung fibroblasts Wi-38 and MRC-5. The death of CAP-exposed cancer and healthy cells under semi-selective conditions was caspase-dependent. We showed that there was an accumulation of lysosomes in the treated cells. The increased activity of lysosomal protease Cathepsin D, the transcriptional upregulation of autophagy-related MAPLC3B gene in cancer cells and the changes in autophagy-related proteins may have indicated the activation of autophagy. The addition of the autophagy inhibitor chloroquine (CQ) after the CAP jet treatment increased the death of A549 cancer cells in a synergistic manner and showed a low effect on the viability of CAP-treated Wi-38 cells. Downregulation of Drp1 mitochondrial protein and upregulation of PINK1 protein in CAP + CQ treated cells indicated that CQ increased the CAP-dependent destabilization of mitochondria. We concluded that CAP weakly activated pro-survival autophagy in irradiated cells, and CQ promoted CAP-dependent cell death due to the destabilization of autophagosomes formation and mitochondria homeostasis. To summarize, the combination of CAP treatment with CQ could be useful for the development of cold plasma-based antitumor approaches for clinical application.

Insight into the impacts of Jinhua ham processing conditions on cathepsin B activity and conformation changes based on molecular simulation

To produce the high quality products and improve the stability of Jinhua hams, the activities of cathepsins and their correlation with processing parameters were explored. The effect of heat treatment on the structure of cathepsin B, together with potential of hydrogen and NaCl concentration, were investigated. At 45 °C, the activity of cathepsin B was maximum and it decreased rapidly at high temperature. The decrease on sulfhydryl group content indicated that the enzyme activity varied due to the breaking of disulfide bond in molecule. Infrared spectroscopy indicated that the secondary structure of cathepsin B had altered. Meanwhile, the root mean squared deviation and the root mean square fluctuation curves reflected that the cathepsin B vitality was in a decline under high temperature. In addition, in the presence of 0.6 mol ⋅ L−1 NaCl, lots of conformational changes were evidenced. Under acidic conditions (pH = 6), there were more formed hydrogen bonds, and the hydrogen bonding interactions became stronger. Simulation results contributed to providing an important theoretical basis for understanding the roles of processing parameters on the quality control of Jinhua ham.

Effects of temperature on cathepsin B, cathepsin D and acid phosphatase during embryo development of the hard tick Haemaphysalis longicornis.

The effects of temperature on the expression patterns and enzyme activity of cathepsin B (HlCatB), cathepsin D (HlCatD) and acid phosphatase (HlACP) during the embryo development of Haemaphysalis longicornis (bisexual population) were investigated in this study. Eggs were exposed to 20°C (low temperature), 26°C (normal temperature), and 30°C (high temperature) immediately after laying, and collected on odd days of embryo development to measure HlCatB, HlCatD and HlACP gene expression using quantitative real-time PCR, as well as three enzyme activities using spectrophotometry. Then the associations between mRNA expression levels of three enzymes and their enzyme activities were assessed. Compared with normal temperature, the mRNA expression peaks of HlCatB were higher and appeared later at low and high temperatures and the activity of HlCatB increased on most days of embryonic development at high temperature. As for HlCatD, the expression peak appeared later at low temperature, but earlier at high temperature. The activity peaks of HlCatD were lower and appeared earlier at low and high temperatures. As for HlACP, the expression peak was higher and appeared later at low temperature, whereas it formed no prominent peak at high temperature. The activity peak of HlACP was higher at low temperature, but lower at high temperature. The linear regression analysis showed that activities of three enzymes were associated with their mRNA expression levels (P < 0.05). Three enzymes are involved in the embryo adaptation to temperature stress. Moreover, the mRNA expression level may be another factor affecting its enzyme activity.

Lack of L-type amino acid transporter 2 in murine thyroid tissue induces autophagy

Proteolytic cleavage of thyroglobulin (Tg) for thyroid hormone (TH) liberation is followed by TH release from thyroid follicles into the circulation, enabled by TH transporters. The existence of a functional link between Tg-processing cathepsin proteases and TH transporters has been shown to be independent of the hypothalamus-pituitary-thyroid axis. Thus, lack of cathepsin K, combined with genetic defects in the TH transporters Mct8 and Mct10, that is the Ctsk-/-/Mct8-/y/Mct10-/- genotype, results in persistent Tg proteolysis due to autophagy induction. Because amino acid transport by L-type amino acid transporter 2 (Lat2) has been described to regulate autophagy, we asked whether Lat2 availability is affected in Ctsk-/-/Mct8-/y/Mct10-/- thyroid glands. Our data revealed that while mRNA amounts and subcellular localization of Lat2 remained unaltered in thyroid tissue of Ctsk-/-/Mct8-/y/Mct10-/- mice in comparison to WT controls, the Lat2 protein amounts were significantly reduced. These data suggest a direct link between Lat2 function and autophagy induction in Ctsk-/-/Mct8-/y/Mct10-/- mice. Indeed, thyroid tissue of Lat2-/- mice showed enhanced endo-lysosomal cathepsin activities, increased autophagosome formation, and enhanced autophagic flux. Collectively, these results suggest a mechanistic link between insufficient Lat2 protein function and autophagy induction in the thyroid gland of male mice.

Apoptosis Inducing Factors Involved in the Changes of Flesh Quality in Postmortem Grass Carp (Ctenopharyngodon idella) Muscle.

Alterations of apoptosis have notable influences on flesh quality, but the mechanism is still unclear. Thus, apoptotic behaviors and related triggering mechanisms need to be explored. Fish muscle was prepared and stored at 4 °C for 0, 24, 48, 72, 96, and 120 h for apoptosis analysis. Results showed that positive apoptotic nuclei were positively correlated with drop loss and negatively correlated with shear force and water holding capacity (p < 0.05). Results showed that the triggering apoptotic mechanisms were involved with enhanced transcriptional levels of caspase-2, 3, 7, 8, and 9 through mitochondria and death receptor pathways in the muscle of grass carp. The decreased ATP content, changed cytochrome c redox state, increased protein levels of HSP27 and HSP 90, and enhanced activity of cathepsin (B, L, and D), calpain, and serine proteinase were involved in apoptosis activations. Results indicated that caspases, energy metabolism, cytochrome c redox state, heat shock protein expressions, and protease activities played critical roles in apoptosis alterations in carp muscle during refrigerated storage.

Loss of TMEM106B exacerbates C9ALS/FTD DPR pathology by disrupting autophagosome maturation.

Disruption to protein homeostasis caused by lysosomal dysfunction and associated impairment of autophagy is a prominent pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The most common genetic cause of ALS/FTD is a G4C2 hexanucleotide repeat expansion in C9orf72 (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 repeat transcripts gives rise to dipeptide repeat (DPR) proteins that have been shown to be toxic and may contribute to disease etiology. Genetic variants in TMEM106B have been associated with frontotemporal lobar degeneration with TDP-43 pathology and disease progression in C9ALS/FTD. TMEM106B encodes a lysosomal transmembrane protein of unknown function that is involved in various aspects of lysosomal biology. How TMEM106B variants affect C9ALS/FTD is not well understood but has been linked to changes in TMEM106B protein levels. Here, we investigated TMEM106B function in the context of C9ALS/FTD DPR pathology. We report that knockdown of TMEM106B expression exacerbates the accumulation of C9ALS/FTD-associated cytotoxic DPR proteins in cell models expressing RAN-translated or AUG-driven DPRs as well as in C9ALS/FTD-derived iAstrocytes with an endogenous G4C2 expansion by impairing autophagy. Loss of TMEM106B caused a block late in autophagy by disrupting autophagosome to autolysosome maturation which coincided with impaired lysosomal acidification, reduced cathepsin activity, and juxtanuclear clustering of lysosomes. Lysosomal clustering required Rab7A and coincided with reduced Arl8b-mediated anterograde transport of lysosomes to the cell periphery. Increasing Arl8b activity in TMEM106B-deficient cells not only restored the distribution of lysosomes, but also fully rescued autophagy and DPR protein accumulation. Thus, we identified a novel function of TMEM106B in autophagosome maturation via Arl8b. Our findings indicate that TMEM106B variants may modify C9ALS/FTD by regulating autophagic clearance of DPR proteins. Caution should therefore be taken when considering modifying TMEM106B expression levels as a therapeutic approach in ALS/FTD.

Effects of cold atmospheric plasma on endogenous enzyme activity and muscle protein oxidation in Trachinotus ovatus

In this study, we investigated the effects of cold atmospheric plasma (CAP) technology on endogenous enzyme characteristics and muscle protein properties of the golden pomfret (Trachinotus ovatus) under different treatment power and time conditions. Results showed that the enzymatic activity of cathepsin B, L, and calpain in crude protease extracts (CPE) decreased significantly as the treatment power and treatment time of CAP increased (p < 0.05). Oxidative degradation of the CPE after exposure to CAP resulted in significant changes in the structure, total sulfhydryl, and carbonyl content of the CPE (p < 0.05). CAP of an appropriate intensity resulted in significant improvements in the color parameters, hydration properties, and textural property parameters of muscle proteins (p < 0.05). These results suggest that CAP, as a non-thermophysical modification technique, can inhibit the activity of endogenous enzymes as well as alter the protein function in food.

Retromer‐dependent mechanisms of tau dysregulation in Down syndrome

AbstractBackgroundDeficiencies in the retromer complex are reported in brains of Down syndrome subjects, and retromer protein levels inversely correlate with amyloidosis. The complex is also proposed to influence tau levels, however, the relationship between tau neuropathology and retromer deficiency in Down syndrome remains unclear. In the current study, we examine the relationship between pathological tau and retromer deficiency and explore potential mechanisms involved.MethodsPost‐mortem brain tissues from Down syndrome and control subjects were evaluated for retromer proteins, pathological tau and proteins relevant to its post‐translational modification. To explore potential mechanisms of retromer‐dependent effects on tau, neuro‐2A cells overexpressing human tau were treated with the pharmacological chaperone, TPT‐172, with or without the CDK5 inhibitor, roscovitine.ResultsPhosphorylated epitopes and insoluble forms tau were elevated in brain tissues of subjects with Down syndrome and inversely correlated with retromer proteins and the activity of cathepsin D. In vitro pharmacological stabilization of the retromer complex with TPT‐172 resulted in decreased levels of phosphorylated tau and the CDK5 activating protein, p25. The effect on tau phosphorylation was neutralized by incubation with the CDK5 inhibitor, roscovitine. Furthermore, CDK5‐p25 immunoprecipitated from cells treated with TPT‐172 caused significantly less histone phosphorylation, indicating that the treatment reduced CDK5 activity.ConclusionWe conclude that in Down syndrome deficiency of retromer proteins is involved in the development of tau neuropathology. We propose that retromer dysfunction contributes to the development of tau pathology by decreasing the degradative lysosomal pathway and by increasing phosphorylation via CDK5. Because pharmacological stabilization of the retromer complex decreases tau phosphorylation, we propose this could represent a novel therapy against tau pathology in Down syndrome.

Human β-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L.

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer's disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer's disease-like neuropathological changes in middle-aged mice. Then, we examined whether human β-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 μM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 μM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.

Cystatin B increases autophagic flux by sustaining proteolytic activity of cathepsin B and fuels glycolysis in pancreatic cancer: CSTB orchestrates autophagy and glycolysis in PDAC.

Both autophagy and glycolysis are essential for pancreatic ductal adenocarcinoma (PDAC) survival due to desmoplasia. We investigated whether targeting a hub gene which participates in both processes could be an efficient strategy for PDAC treatment. The expression pattern of glycolysis signatures (GS) and autophagy signatures (AS) and their correlation with cystatin B (CSTB) in PDAC were analysed. It was discovered how CSTB affected the growth, glycolysis, and autophagy of PDAC cells. We assessed competitive binding to cathepsin B (CTSB) between CSTB and cystatin C (CSTC) via immunoprecipitation (IP) and immunofluorescence (IF). Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays were used to unveil the mechanism underlying CSTB upregulation. The expression pattern of CSTB was examined in clinical samples and KrasG12D/+, Trp53R172H/+, Pdx1-Cre (KPC) mice. GS and AS were enriched and closely associated in PDAC tissues. CSTB increased autophagic flux and provided substrates for glycolysis. CSTB knockdown attenuated the proliferation of PDAC cells and patient-derived xenografts. The liquid chromatography-tandem mass spectrometry assay indicated CSTB interacted with CTSB and contributed to the proteolytic activity of CTSB in lysosomes. IF and IP assays demonstrated that CSTB competed with CSTC to bind to CTSB. Mutation of the key sites of CSTB abolished the interaction between CSTB and CTSB. CSTB was highly expressed in PDAC due to H3K27acetylation and SP1 expression. High expression of CSTB in PDAC was observed in tissue microarray and patients' serum samples. Our work demonstrated the tumorigenic roles of autophagy and glycolysis in PDAC. CSTB is a key role in orchestrating these processes to ensure energy supply of PDAC cells.

Loss-of-function mutations in CST6 cause dry skin, desquamation and abnormal keratosis without hypotrichosis.

Cystatin M/E (encoded by the CST6 gene) is a cysteine protease inhibitor, that exerts regulatory and protective effects against uncontrolled proteolysis mainly by directly regulating cathepsin V, cathepsin L, and legumain activities. Previous studies have suggested that CST6 may exert a regulatory role in epidermal differentiation and hair follicle formation by inhibiting the activity of respective cognate target proteases. However, until recently, studies have revealed that loss- or gain-of-function of the CST6 gene causes dry skin with hypotrichosis in humans. Here, we reported two siblings of Chinese origin with dry skin, desquamation and abnormal keratosis without hypotrichosis. By applying whole-exome sequencing, we identified homozygous loss-of-function mutation c.251G > A (p.Gly84Asp) in the CST6 gene as the underlying genetic cause. Further fluorimetric enzyme assays demonstrated the mutant cystatin M/E protein lost its inhibitory function on the protease activity of cathepsins. Moreover, the corresponding mutation in mice resulted in excessive cornification, desquamation, impaired skin barrier function, and abnormal proliferation and differentiation of keratinocytes. In conclusion, the homozygous missense mutation c.251G > A in CST6 gene resulted in dry skin, desquamation, as well as abnormal keratosis of the skin, promoting our understanding of the role of protease-antiprotease balance in human skin disorders.

An altered transcriptome underlies cln5-deficiency phenotypes in Dictyostelium discoideum.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of neurodegenerative lysosomal storage diseases that affect all ages and ethnicities globally. Previous research showed that CLN5 participates in a variety of cellular processes. However, the precise function of CLN5 in the cell and the pathway(s) regulating its function are not well understood. In the model organism Dictyostelium discoideum, loss of the CLN5 homolog, cln5, impacts various cellular and developmental processes including cell proliferation, cytokinesis, aggregation, cell adhesion, and terminal differentiation. In this study, we used comparative transcriptomics to identify differentially expressed genes underlying cln5-deficiency phenotypes during growth and the early stages of multicellular development. During growth, genes associated with protein ubiquitination/deubiquitination, cell cycle progression, and proteasomal degradation were affected, while genes linked to protein and carbohydrate catabolism were affected during early development. We followed up this analysis by showing that loss of cln5 alters the intracellular and extracellular amounts of proliferation repressors during growth and increases the extracellular amount of conditioned medium factor, which regulates cAMP signalling during the early stages of development. Additionally, cln5 - cells displayed increased intracellular and extracellular amounts of discoidin, which is involved in cell-substrate adhesion and migration. Previous work in mammalian models reported altered lysosomal enzyme activity due to mutation or loss of CLN5. Here, we detected altered intracellular activities of various carbohydrate enzymes and cathepsins during cln5 - growth and starvation. Notably, cln5 - cells displayed reduced β-hexosaminidase activity, which aligns with previous work showing that D. discoideum Cln5 and human CLN5 can cleave the substrate acted upon by β-hexosaminidase. Finally, consistent with the differential expression of genes associated with proteasomal degradation in cln5 - cells, we also observed elevated amounts of a proteasome subunit and reduced proteasome 20S activity during cln5 - growth and starvation. Overall, this study reveals the impact of cln5-deficiency on gene expression in D. discoideum, provides insight on the genes and proteins that play a role in regulating Cln5-dependent processes, and sheds light on the molecular mechanisms underlying CLN5 disease.

Effects of capture-related stress and pre-freezing holding in refrigerated sea water (RSW) on the muscle quality and storage stability of Atlantic mackerel (Scomber scombrus) during subsequent frozen storage

Crowded (stressed) and unstressed Atlantic mackerel with or without pre-freezing holding in refrigerated sea water (RSW) were stored at −19 °C for ∼12 months and analysed for nucleotide degradation (K value), muscle pH, water holding capacity (WHC), fillet firmness, cathepsin B/L like activity, lipid oxidation and fillet colour. The frozen storage showed the largest and most consistent direct effects on the quality metrics leading to increased lipid oxidation, discolouration (yellowing) and reduction on WHC and cathepsin activity. RSW treatment promoted nucleotide degradation and reduced WHC and fillet firmness in interaction with frozen storage and affected fillet colour lightness and saturation. Although showing only marginal main effects, crowding stress modified WHC, cathepsin activity and fillet firmness and colour through significant interactions with the frozen storage and RSW treatment. Further studies with larger sample sizes would be needed to elucidate their complex effects and interactions on the quality and storage stability of mackerel.

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli.

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition. The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (≥ 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Effects of Primary Processing and Pre-Salting of Baltic Herring on Contribution of Digestive Proteases in Marinating Process

The low-technological quality of herring caught during the feeding season makes it impossible to achieve full ripeness of the meat in marinades. One solution may be to assist ripening using herring digestive tract proteases. Therefore, whole herring, headed herring and fillets were marinated for 2–14 days using the German (direct) and Danish (pre-salted) methods. The results showed that the mass of marinades from fillets was lower than from herring with intestines and correlated strongly with salt concentration in the Danish method, in contrast to the German method. Marinades from whole and headed herring had significantly higher trypsin, chymotrypsin, carboxypeptidase-A and cathepsin activities than marinated fillets. The herring marinated with viscera had 2–3 times higher non-protein nitrogen, peptide and amino acid fractions, as well as ripened 3 days faster than the marinated fillets. After 2 weeks of marinating, the fillets did not achieve full ripeness of the meat, unlike marinades made from whole and headed herring. The pre-salting stage in the Danish method significantly reduced cathepsin D activity by the tenth day of marinating, which was compensated by digestive proteases only in the case of whole or headed herring. The digestive proteases activity in the fillets was too low to achieve the same effect. Sensory evaluation of texture and hardness-TPA correlated strongly with several proteases in whole herring marinades, in contrast to a weak correlation with only one protease when marinating fillets. Marinating with intestines makes it possible to produce marinades faster, more efficiently and with higher sensory quality from herring of low-technological quality.

Protecting glomerulus: role of angiotensin-II type 2 receptor.

Podocyte injury due to either drug, toxin, infection, or metabolic abnormality is a great concern as it increases the risk of developing focal segmental glomerulosclerosis (FSGS) and proteinuric kidney diseases. The direct podocyte injury due to doxorubicin is associated with an increase in proinflammatory cytokines and induction of cathepsin L. The increased activity of cathepsin L in turn may degrade the glomerular slit diaphragm resulting in proteinuric kidney injury. The angiotensin-II type 2 receptor (AT2R) has earlier been reported to be associated with the preservation of slit diaphragm proteins and prevention of proteinuria. Recent in vivo findings by Zhang and colleagues further support the anti-proteinuric role of AT2R in preventing podocyte injury via down-regulating cytokines ccl2, and hence, cathepsin L, thereby, limiting the progression of FSGS.

Effects of ultra high pressure-magnetic field treatment on protein properties and quality characteristics of stored shrimp (LitopenaeusVannamei)

The preservation of ultra high pressure (UHP) and magnetic field (MF) technologies on food have been reported, but the effect of combined treatment (UHP-MF) on aquatic product quality is rarely explored. The purposes of this study were to investigate the effects of UHP-MF treatment on protein properties and quality deterioration of stored Litopenaeus Vannamei. The vacuum-packed shrimps were treated at 200 and 300 MPa for 5 min, then stored in 5 mT MF for 12 days. Results showed the hardness, springiness, chewiness, shear force, L*, a*, b*, and W of muscle were improved by UHP, whereas the texture softening, a* and b* increase, water holding capacity were inhibited during MF storage. On the 12th day, compared with CON group, the cathepsin B, D, H, L activities, TBARS, carbonyl and dityrosine content in UHP300-MF group were reduced by 46.67%, 27.26%, 43.10%, 52.00%, 41.30%, 32.86%, and 30.42%, respectively. Furthermore, UHP-MF treatment slow down the accumulation of surface hydrophobicity, myofibril fragmentation index, and trichloroacetic acid-soluble peptide content. Generally, UHP-MF treatment inhibited lipid oxidation, weakened cathepsin activities, delayed protein oxidation and degradation of stored shrimp, retarded quality deterioration and prolonged shelf life of stored shrimp.